PROJECT SUMMARY: The regulation of protein synthesis plays a major role in controlling gene expression and consequently cell growth, proliferation and differentiation. Rates of protein synthesis are altered in response to nutrient availability, hormones and growth factors and therefore are directly coupled with cell cycle progression. Our long-term goal is to understand the molecular mechanism and regulation of mammalian protein synthesis initiation. The recruitment of an mRNA to the ribosome requires the formation and maintenance of fundamental interactions between eukaryotic initiation factors (eIFs), the mRNA and the ribosome. The large eIF3 complex binds directly to the 40S ribosomal subunit, promoting mRNA recruitment through an insulin-regulated interaction with the eIF4G subunit of the cap-binding complex (eIF4E, eIF4G and eIF4A). The hypothesis is that specific interactions between the cap- binding complex, eIF3 and the 40S subunit are essential to recruit mRNA to the ribosome, promote helicase activity, and induce conformation rearrangements in the ribosome to initiate protein synthesis. We will test this hypothesis using biochemical and biophysical approaches to study the structural and kinetic basis of initiation factor functions in promoting important intermediates in the protein synthesis initiation pathway. The Specific Aims are: I. Characterize the association of eIF4G with eIF3 and the 40S subunit. We will determine the consensus sequence in eIF4G that binds to eIF3. Important amino acids will be identified using the fluorescence polarization assay we have established together with site-directed mutagenesis of eIF4G. We will exploit these mutants to determine how this interaction regulates mRNA recruitment and protein synthesis in vivo. We will also reveal how a novel RNA binding region contributes to the stability of eIF4G with the eIF3-40S complex in vitro and in vivo. Subunits of eIF3 that interact with eIF4G will be identified using site-directed crosslinking. The role of identified eIF3 subunits will be confirmed by competition assays in vitro and in vivo. II. Elucidate how initiation factors promote structural changes in the 40S subunit enabling mRNA recruitment and AUG recognition. The mechanism by which the hepatitis c virus (HCV) internal ribosome entry site (IRES) functionally replaces the cap-binding complex in mRNA recruitment to the 40S subunit will be identified. Using a site-directed RNA cleavage assay we will determine the contribution that each initiation factor makes in promoting structural rearrangement of the 40S subunit during intermediates of the initiation pathway. This will provide a structural basis for initiation factor functions in mRNA recruitment. III. Determine how human ribosomes scan along the 5 untranslated region (5UTR) to locate the AUG codon. We will establish a fluorescent kinetic RNA unwinding assay to determine how initiation factors contribute to the migration rate of the 40S subunit through secondary structure. The helicase activity of initiation factors and the 40S subunit during scanning will be visualized using a single-molecule assay, in collaboration with Steven Block (Stanford).